Human cytomegalovirus is the prototypical member of the beta-herpes virus family. Epidemiological studies have shown that human cytomegalovirus infection is widespread. In healthy individuals infection is generally asymptomatic, but the virus can cause serious disease in people with immature or compromised immune systems. It is the leading infectious disease cause of birth defects and a life-threatening adventitious agent in transplant recipients. The long-term objective of this research program is to elucidate the function of human cytomegalovirus genes that regulate the interaction of the virus with its host cell and thereby control viral replication and pathogenesis. This proposal is designed to contribute to the understanding of several of the G protein-coupled receptors encoded by HCMV. One of the receptors to be studied is the product of the US28 gene. Preliminary studies have shown that a mutant human cytomegalovirus lacking the US28 coding region exhibits an accelerated pattern of gene expression after infection of G0 fibroblasts; early and late mRNAs accumulate long before they are detected in wild-type virus-infected fibroblasts. In the absence of the US28 receptor, the time-dependent cascade of immediate-early, early and late gene expression observed in wild-type virus infections is disrupted. The first specific aim seeks to characterize the effects of HCMV-coded pUS28 on the transcriptional cascade within multiple infected cell types and explore the mechanism of pUS28 action. The second G protein-coupled receptor to be studied is the product of the UL78 gene. The murine cytomegalovirus homologue of this gene is packaged into virions, and, after the receptor is delivered to cells by fusion of the virion envelope with the plasma membrane, it facilitates activation of the viral m123 immediate-early gene. In the second specific aim human cytomegalovirus mutants that do not express the UL78 product will be characterized in multiple cell types to explore the function of that receptor. This project will include analysis of a laboratory strain (AD169) and two clinical isolates (VR1814, FIX; and PH) of human cytomegalovirus. Viral replication will be studied in fibroblasts, the cell commonly used to propagate HCMV; aortic endothelial and smooth muscle cells, which are relevant to the possible role of HCMV in atherosclerosis; retinal pigmented epithelial cells, which are infected in viral retinitis; and macrophages, which facilitate virus spread.